Agilent Human 14
Multiple Affinity
Removal System Spin
Cartridges for the
Depletion of
High-Abundant
Proteins from Human
Proteomic Samples
Instructions
Second edition
October 2008
General Information
Introduction
The Agilent Human 14 Multiple Affinity Removal
System comprises a family of immunodepletion
products based on affinity interactions and optimized buffers for sample loading, washing, eluting,
and regenerating. This spin cartridge is specifically
designed to remove 14 high-abundant proteins from
human biological fluids such as plasma, serum, and
cerebral spinal fluid (CSF). This technology enables
removal of albumin, IgG, antitrypsin, IgA, transferrin,
haptoglobin, fibrinogen, alpha2-macroglobulin,
alpha1-acid glycoprotein, IgM, apolipoprotein AI,
apolipoprotein AII, complement C3, and transthyretin with a single device. The targeted high-abundant
proteins are simultaneously removed when crude
biological samples are passed through the cartridge.
Selective immunodepletion provides an enriched
pool of low-abundant proteins for downstream proteomics analysis.
Specific removal of 14 high-abundant proteins
depletes approximately 94% of total protein mass
from human serum/plasma. The low-abundant proteins in the flow-through fractions can be studied.
Removal of high-abundant proteins enables
improved resolution and dynamic range for
one-dimensional gel electrophoresis (1DGE),
two-dimensional gel electrophoresis (2DGE), and
liquid chromatography/mass spectrometry
Instructions
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(LC/MS). The collected flow-through fractions may
need to be concentrated dependent upon the downstream applications.
Instructions
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Human 14 Multiple Affinity Removal
System
The Agilent Human 14 Multiple Affinity Removal
System is specially designed to allow for close study
of low-abundant proteins present in flow-through
fractions (see Figure 1).
H H
L H L
H L LH
L H
H LH L H
Apply Crude Human
Serum/Plasma
Human Proteins Depleted:
Albumin, IgG, Antitrypsin,
IgA, Transferrin, Haptoglobin,
Fibrinogen, Alpha2-Macroglobulin,
Alpha1-Acid Glycoprotein, IgM,
Apolipoprotein AI, Apolipoprotein AII,
Complement C3, Transthyretin
L L
L LL LL
HH
H H
H
H HH
Fraction #1
Low-Abundant
Proteins
Fraction #2
High-Abundant
Proteins
Figure 1 The Multiple Affinity Removal System.
Instructions
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Product Description
The Agilent Human 14 Multiple Affinity Removal
System Spin Cartridge and its accessories are
shown in Table 1.
Instructions
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Table 1 Agilent Human 14 Multiple Affinity
Removal System Spin Cartridge and
Starter Reagent Kit
Product no. Product name Product description
5188-6560 0.45-mL affinity, Removes human albumin,
spin cartridge, IgG, antitrypsin, IgA, trans1 each
ferrin, haptoglobin, fibrinogen, alpha2-macroglobulin, alpha1-acid glycoprotein, IgM, apolipoprotein
AI, apolipoprotein AII,
complement C3, and transthyretin
5185-5987 Buffer A, 1 L
Ready-to-use, optimized
buffer for loading, washing, and equilibrating cartridge
5185-5988 Buffer B, 1 L
Ready-to-use, optimized
buffer for elution of bound
proteins from cartridge
5185-5990 Spin filters
For sample cleanup before
0.22 µm,
loading cartridge
1 pack of 25
5185-5991 Protein spin
For concentrating
concentrators, flow-through fractions
5kDa MWCO,
4 mL, 1 pack
of 25
5188-5249 Luer-Lock
Allows attachment of
adapters,
Luer-Lock syringes to spin
1 pack of 2
cartridge
Instructions
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Product no. Product name Product description
5188-5250 5-mL plastic
For washing, eluting, and
Luer-Lock
re-equilibrating buffers
syringes,
through spin cartridge
1 pack of 2
5188-5251 1.5-mL screwtop Eppendorf-style tubes
microtube,
used for collecting frac1 pack of 100 tions from spin cartridge
5188-5252 Spin cartridge Extra caps and plugs for
screw caps
sealing the top and bottom
and plugs,
of affinity spin cartridges
1 pack of 6 each
5188-5253 Teflon LuerFor transferring solutions
Lock needles, with Luer-Lock Syringes
1 pack of 10
5188-5254 Starter Reagent Kit for Spin Cartridges*
Buffer A: 1 L
Buffer B: 1 L
Spin filters 0.22 µm: 2 packs of 25
Protein concentrators: 1 pack of 25
Luer-Lock adapters: 1 pack of 2
5-mL plastic Luer-Lock syringes: 1 pack of 2
1.5-mL microtubes: 6 packs of 100
Spin cartridge extra caps and plugs, 1 pack
of 6 each
Teflon Luer-Lock needles, 1 pack of 10
* Under normal conditions, the kit should last for approximately 200 spin cartridge uses.
Instructions
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N O TE
Instructions
For higher capacity Multiple Affinity Removal
System Devices, and if automated immunodepletion is needed, refer to Human 14 Multiple Affinity Removal System Columns (part number
5188-6557 - 4.6 x 50 mm, 5188-6558 - 4.6 x 100
mm, 5188-6559 - 10 x 100 mm) for use with HPLC
instrumentation.
See www.agilent.com/chem/bioreagents for
more information and links to mouse protein
removal devices.
8
Full Protocol for Human 14 Multiple
Affinity Removal System Spin Cartridge
(Cartridge capacity: 8–10 µL human serum/plasma*)
During use, never let the spin cartridge frits or
resin bed run dry. If this happens, see Recommendations section for cartridge rewetting procedure.
N O TE
Additional Materials Required
• Microcentrifuge with adjustable centrifugal force
(capable of spinning at 100 x g) and timer such as
the Eppendorf Model 5415D
• 50-mL vessels (for example, polypropylene tubes)
to hold working quantities of Buffers A and B during procedure
• Adjustable pipettes for delivering up to 400-µL aliquots
* Capacity testing was done with pooled normal plasma
from Rockland Immunochemicals (D519-04).
Consult certificate of analysis to verify your capacity.
It is the user's responsibility to adjust loading volume to
compensate for the fact that the concentrations of some
high-abundant proteins can vary widely depending on the
sample origin (source). Several serum/plasma proteins
such as alpha1-antitrypsin, haptoglobin, fibrinogen, IgG,
and alpha1-acid glycoprotein rise several folds in
response to stress, infection, inflammation, or tissue
necrosis and are known as acute phase reactants (Henry,
J.B. 1996 - Clinical Diagnosis and Management by Laboratory Methods).
Instructions
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• Transfer pipettes
• 1.5-mL screw-top microtubes/Eppendorf-style
tubes (collection tubes, part number 5188-5251)
• Luer-Lock adapters (part number 5188-5249)
• 5-mL plastic Luer-Lock syringes (part number
5188-5250)
• Buffer A, 1 L (part number 5185-5987)
• Buffer B, 1 L (part number 5185-5988)
Material Preparation
• Fill two 50-mL vessels with appropriate amounts
of Buffers A and B to use throughout the procedure for the specific number of samples you wish
to process (approximately 5-mL Buffer A and
2-mL Buffer B per each 8–10 µL serum/
plasma sample).
• Label two 5-mL Luer-Lock syringes with "A" and
"B" for using later in the procedure during the
cartridge elution and re-equilibration steps.
N O TE
Instructions
Remove Agilent Human 14 Multiple Affinity
Removal System Spin Cartridge from refrigerator
and allow cartridge to equilibrate to room temperature before use.
10
Procedure
1 Dilute and filter sample. Prepare the sample by
diluting 8–10 µL of human serum/plasma* with
Buffer A to a final volume of 200 µL. For example:
if the recommended cartridge loading capacity
on the certificate is 10 µL of serum/plasma,
dilute 10 µL of serum/plasma with 190-µL
Buffer A† to a final volume of 200 µL.
If you plan to perform several successive runs on
the cartridge, increase amount of diluted sample
accordingly.
Filter diluted samples through a 0.22-µm spin filter (part number 5185-5990) to prevent clogging
of spin cartridge frits.
2 Prepare spin cartridge. Remove cartridge cap
and plug. Attach Luer-Lock adapter to spin cartridge, draw 4 mL of Buffer A into syringe and
then attach it to Luer-Lock on spin cartridge.
Dispense Buffer A through spin cartridge to prepare resin and to remove any trapped air bubbles. With a transfer pipette, remove excess
Buffer A from top of the spin cartridge.
* Protocol may be applied to other human biological fluids
with necessary adjustments in sample volume based on
albumin concentration.
† Addition of protease inhibitors in Buffer A for sample
dilution helps prevent protein degradation.
Instructions
11
3 Apply sample. Remove the Luer-Lock adapter
and place spin cartridge in a screw-top collection
tube and label it "Flow-through 1" or "F1". Add
200 µL of diluted serum/plasma sample to spin
cartridge and centrifuge for 1.0 minute at 100 × g
(or lowest possible setting on centrifuge; see
note below). Cap the spin cartridge loosely or
leave open during centrifugation so the sample
is able to flow. Collect the flow-through fraction
in the collection tube F1. The resin bed and frits
should remain moist, not dry, after centrifugation.
If centrifuge cannot be programmed to 100 x g,
then cartridge capacity may be different for use;
the optimum results for depletion are obtained
when the flow rate is controlled to < 0.2 mL/min.
N O TE
4 Incubate. Remove spin cartridge from centrifuge
and allow to sit for 5 minutes at room temperature.
5 Wash and collect flow-through fraction F1.
Add 400 µL of Buffer A to the top of the resin bed
and centrifuge for 2.5 minutes at 100 × g. Collect
the flow-through fraction into the same F1 collection tube.
6 Wash and collect additional flow-through fraction F2. Place spin cartridge into a new collection tube labeled "Flow-through 2" or "F2". Add
400 µL of Buffer A to the top of the resin bed and
Instructions
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centrifuge for 2.5 minutes at 100 × g. Collect the
flow-through fraction into the F2 collection tube.
7 Prepare for elution. Remove the spin cartridge
from the F2 collection tube and attach Luer-Lock
adapter to the cartridge top.
8 Elute bound fraction. Fill a 5-mL plastic
Luer-Lock syringe (labeled "B") with 2.5 mL of
Buffer B and attach to the spin cartridge via the
Luer-Lock adapter. Elute bound high-abundant
proteins into a new collection tube by slowly
pushing Buffer B through the spin cartridge.
Save the bound fraction for analysis if desired, or
discard it. Do not push air through the spin cartridge and do not allow the resin bed or frits to
run dry.
If the meniscus of Buffer B does not reach the
top frit after depressing the syringe plunger completely, remove syringe and draw the plunger
back to the 1-mL mark with air and reattach to
the cartridge. Use the air in the syringe as positive pressure to push Buffer B through until the
meniscus of Buffer B reaches the top frit.
N O TE
9 Re-equilibrate. Remove Buffer B syringe and
attach a 5-mL syringe (labeled "A") containing
5 mL of Buffer A to the spin cartridge. Re-equilibrate the spin cartridge by slowly pushing
Buffer A through the resin bed. Do not allow the
resin bed or frits to run dry by leaving a small aliquot of buffer on the top of the frit. The spin
Instructions
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cartridge is ready for the next sample. For storage, leave the resin bed wet with Buffer A, and
leave a layer of Buffer A above the top frit. Recap
both ends of the spin cartridge tightly.
Be careful when placing the plug in lower end of
the spin cartridge; it must not displace or puncture the lower frit. Store spin cartridge in Buffer
A in a refrigerator at 2–8 °C (35–46 °F). DO NOT
FREEZE THE SPIN CARTRIDGE.
N O TE
10 Analyze. Analyze separately or combine
flow-through fractions F1 and F2 containing the
low-abundant proteins. For 1D-SDS-PAGE, an aliquot of the flow-through fraction may be used
directly. For IEF, 2DGE, and MS analysis of the
flow-through fraction, it is necessary to buffer
exchange/desalt to an appropriate buffer. The
5KDa MWCO spin concentrators (part number
5185-5991) may be used for buffer exchange and
concentration. Alternatively, for automated
desalting and concentration, the Agilent
mRP-C18 column (part number 5188-5231) may
be used according to published methods (Agilent
Technologies, publication 5989-2506EN).
N O TE
Instructions
For maximum recovery of the flow-through
protein combine and concentrate fractions F1
and F2.
14
Recommendations
• Sample dilution
It is not recommended to load crude serum or
plasma directly onto the spin cartridge. Follow
instructions for serum/plasma dilution. Addition
of protease inhibitors in Buffer A for sample dilution helps prevent protein degradation.
• Sample cleanup
Human serum or plasma may contain particulate
materials that can be removed by a quick spin
using a 0.22-µm spin filter (part number
5185-5990).
• Sample concentration
For further downstream proteomic analysis
(SDS-PAGE or LC/MS), combine flow-through
fractions F1 and F2 and concentrate the samples.
Spin concentrators with 5 KDa MWCO (part number 5185-5991) can be used to concentrate proteins before analysis. Alternatively, for automated
desalting and concentration, the Agilent
mRP-C18 column (part number 5188-5231) may
be used according to published methods (Agilent
Technologies, publication 5989-2506EN).
• Bound fraction analysis
If you will be analyzing the bound fraction, perform a buffer exchange to phosphate-buffered
saline (PBS) or to another buffer compatible with
your analysis. Buffer B contains compounds that
may interfere with some protein assays.
Instructions
15
• Cartridge rewetting
If the resin bed becomes dry during cartridge centrifugation or syringe elution, attach a syringe
with Buffer A to the spin cartridge via the
Luer-Lock adapter and re-equilibrate the cartridge
by passing Buffer A through it. This should not
affect the spin cartridge performance.
• Spin cartridge performance
Human 14 Multiple Affinity Removal System Spin
Cartridges should perform reproducibly for more
than 200 runs under proper conditions. Buffers A
and B are optimized to support cartridge performance and longevity. Agilent does not guarantee
spin cartridge performance if other buffers are
used. Do not expose cartridges to organic solvents (like alcohols, acetonitrile, etc.), strong oxidizers, acids, or reducing agents, and other
protein denaturing agents.
• Cartridge storage
After equilibrating Multiple Affinity Removal System Spin Cartridges with Buffer A, always store
them in a refrigerator at 2–8 °C (35–46 °F) when
not in use to minimize loss in cartridge capacity.
• Lyophilization of flow-through fractions
Buffer exchange to a volatile buffer (for example,
ammonium bicarbonate) is recommended prior to
lyophilization due to high salt concentration in
Buffer A.
Instructions
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Troubleshooting
Problem
No flow
Cause
The spin cartridge
may be capped too
tightly during centrifugation.
Bubble under resin
or frits.
Solution
Remove or loosen
cap during centrifugation.
Rewet with Buffer A
(see Recommendations - Cartridge
rewetting).
Incomplete Centrifugation para- Adjust centrifuge
flow
meters need to be force and time to
adjusted
achieve
≤ 0.2-mL/min flow
rates through spin
cartridge during
steps 3 through 5 of
procedure.
No proteins Buffers A and B
Re-equilibrate spin
in bound
reversed
cartridge with
fraction
Buffer A (step 8) and
start over with correct buffer sequence.
Instructions
17
Problem
Cause
BreakExceeding cartridge
through of serum/plasma
highcapacity
abundant
proteins in Serum/plasma proflow-through tein levels may be
fractions F1 unusually high
and F2
Flow rate through
cartridge during
sample loading too
high
Instructions
Solution
Reduce
serum/plasma load
per sample.
Reduce
serum/plasma load
per sample.
Reduce centrifugation force and/or
time during sample
loading to not exceed
0.2 mL/min flow rate.
18
Cartridge Specifications
Part number 5188-6560
Cartridge body material
Frit materials
Cartridge capacity*
Recommended centrifugal
force
Operating temperature
Cartridge packing material
Immobilized ligands
Shipping solution
Shipping temperature
Storage temperature
0.45-mL Multiple Affinity
Removal System Cartridge,
1 each
Polypropylene
Polyethylene with 10-µm
pore size
8–10 µL human
serum/plasma
100 x g
18–25 °C
Affinity resin
Affinity ligands to human
albumin, IgG, antitrypsin,
IgA, transferrin, haptoglobin,
fibrinogen, alpha2-macroglobulin, alpha1-acid glycoprotein, IgM, apolipoprotein
AI, apolipoprotein AII, complement C3, and transthyretin
Buffer A with 0.02% sodium
azide
2–8 °C (35–46 °F)
2–8 °C (35–46 °F)
* For exact cartridge capacity, consult your cartridge certificate of analysis.
Instructions
19
Do not expose cartridges to organic solvents
(like alcohols, acetonitrile, etc.), strong oxidizers, acids, or reducing agents, and other protein denaturing agents.
CAU T ION
For RESEARCH USE ONLY. This product is
NOT TO BE USED AS AN IN-VITRO
DIAGNOSTIC.
WARN IN G
Storage: Store the affinity cartridge at 2–8 °C
(35–46 °F) upon receiving and when not in use. DO
NOT FREEZE THE CARTRIDGE! Store cartridge wetted with Buffer A and with the end-caps tightly
sealed.
Instructions
20
Safety Issues
When preparing biological samples using Agilent
Human 14 Multiple Affinity Removal System Spin
Cartridges, follow general guidelines for handling
biological materials and wear protective eyewear
and gloves.
Related Agilent Products
Other related Agilent products include the following:
5188-6557 Agilent Multiple Affinity Removal System
Column Hu-14, 4.6 X 50 mm, LC column
that depletes 14 high-abundant proteins
(albumin, IgG, IgA, transferrin, haptoglobin, antitrypsin, fibrinogen, alpha2-macroglobulin, alpha1-acid glycoprotein, IgM,
apolipoprotein AI, apolipoprotein AII, complement C3, and transthyretin ) from human
serum/plasma samples, 20 µL serum/
plasma capacity per injection
Instructions
21
5188-6558 Agilent Multiple Affinity Removal System
Column Hu-14, 4.6 X 100 mm, LC column
that depletes 14 high-abundant proteins
(albumin, IgG, IgA, transferrin, haptoglobin, antitrypsin, fibrinogen, alpha2-macroglobulin, alpha1-acid glycoprotein, IgM,
apolipoprotein AI, apolipoprotein AII, comple- ment C3, and transthyretin) from
human serum/plasma samples, 40 µL
serum/plasma capacity per injection
5188-6559 Agilent Multiple Affinity Removal System
Column Hu-14, 10 X 100 mm, LC column
that depletes 14 high-abundant proteins
(albumin, IgG, IgA, transferrin, haptoglobin, antitrypsin, fibrinogen, alpha2-macroglobulin, alpha1-acid glycoprotein, IgM,
apolipoprotein AI, apolipoprotein AII,
complement C3, and transthyretin) from
human serum/plasma samples, 250 µL
serum/plasma capacity per injection
5188-6409 Agilent High Capacity Multiple Affinity
Removal System Column Hu-7HC,
4.6 X 50 mm, LC column that depletes
seven high-abundant proteins (albumin,
IgG, IgA, transferrin, haptoglobin, antitrypsin, and fibrinogen) from human
serum/plasma samples, 30–35 µL
serum/plasma capacity per injection
Instructions
22
5188-6410 Agilent High Capacity Multiple Affinity
Removal System Column Hu-7HC,
4.6 X 100 mm, LC column that depletes
seven high-abundant proteins (albumin,
IgG, IgA, transferrin, haptoglobin, antitrypsin, and fibrinogen) from human
serum/plasma samples, 60–70 µL
serum/plasma capacity per injection
5188-6411 Agilent High Capacity Multiple Affinity
Removal System Column Hu-7HC,
10 X 100 mm, LC column that depletes
seven high-abundant proteins (albumin,
IgG, IgA, transferrin, haptoglobin, antitrypsin, and fibrinogen) from human
serum/plasma samples, 250–300 µL
serum/plasma capacity per injection
5188-6408 Agilent High Capacity Multiple Affinity
Removal System Spin Cartridge Hu-7HC,
spin cartridge that depletes seven
high-abundant proteins (albumin, IgG, IgA,
transferrin, haptoglobin, antitrypsin, and
fibrinogen) from human serum/plasma
samples, 12–14 µL serum/plasma capacity
per use
5188-5332 Agilent High Capacity Multiple Affinity
Removal System Column Hu-6HC,
4.6 X 50 mm, LC column that depletes six
high-abundant proteins (albumin, IgG, IgA,
transferrin, haptoglobin, and antitrypsin)
from human serum/plasma samples,
30–40 µL serum/plasma capacity per injection
Instructions
23
5188-5333 Agilent High Capacity Multiple Affinity
Removal System Column Hu-6HC,
4.6 X 100 mm, LC column that depletes six
high-abundant proteins (albumin, IgG, IgA,
transferrin, haptoglobin, and antitrypsin)
from human serum/plasma samples,
60–80 µL serum/plasma capacity per injection
5188-5336 Agilent High Capacity Multiple Affinity
Removal System Column Hu-6HC,
10 X 100 mm, LC column that depletes six
high-abundant proteins (albumin, IgG, IgA,
transferrin, haptoglobin, and antitrypsin)
from human serum/plasma samples,
300–325 µL serum/plasma capacity per
injection
5188-5341 Agilent High Capacity Multiple Affinity
Removal System Spin Cartridge Hu-6HC,
spin cartridge that depletes six high-abundant proteins (albumin, IgG, IgA, transferrin, haptoglobin, and antitrypsin) from
human serum/plasma samples, 14–16 µL
serum/plasma capacity per use
5185-5984 Multiple Affinity Removal System Column
Hu-6, 4.6 × 50 mm, LC column that
depletes six high-abundant proteins (albumin, IgG, IgA, transferrin, haptoglobin, and
antitrypsin) from human serum/plasma
samples, 15–20 µL serum/plasma capacity
per injection
Instructions
24
5185-5985 Multiple Affinity Removal System Column
Hu-6, 4.6 × 100 mm, LC column that
depletes six high-abundant proteins (albumin, IgG, IgA, transferrin, haptoglobin, and
antitrypsin) from human serum/plasma
samples, 30–40 µL serum/plasma capacity
per injection
5188-2714 Multiple Affinity Removal System Column
Hu-6, 10 × 100 mm, LC column that
depletes six high-abundant proteins (albumin, IgG, IgA, transferrin, haptoglobin, and
antitrypsin) from human serum/plasma
samples, 140–190 µL serum/plasma
capacity per injection
5188-5230 Multiple Affinity Removal System Spin
Cartridge Hu-6, spin cartridge that
depletes six high-abundant proteins (albumin, IgG, IgA, transferrin, haptoglobin, and
antitryp- sin) from human serum/plasma
samples, 7–10 µL serum/plasma capacity
per use
5188-5217 Multiple Affinity Removal System Column
Ms-3, 4.6 × 50 mm, LC column that
depletes three high-abundant proteins
(albumin, IgG, and transferrin) from mouse
serum/plasma samples, 37–50 µL
serum/plasma capacity per injection
5188-5218 Multiple Affinity Removal System Column
Ms-3, 4.6 × 100 mm, LC column that
depletes three high-abundant proteins
(albumin, IgG, and transferrin) from mouse
serum/plasma, 75–100 µL serum/plasma
capacity per injection
Instructions
25
5188-5289 Multiple Affinity Removal System Spin
Cartridge Ms-3, spin cartridge that
depletes three high-abundant proteins
(albumin, IgG, and transferrin) from mouse
serum/plasma samples, 25–30 µL
serum/plasma capacity per use
5185-5986 Multiple Affinity Removal System Reagent
Kit, starter reagent kit containing buffers,
spin filters, and spin concentrators for use
with Multiple Affinity Removal System LC
Columns
5188-5254 Starter Reagent Kit for Spin Cartridges*
Buffer A: 1 L
Buffer B: 1 L
Spin filters 0.22 µm: 2 packs of 25
Protein concentrators: 1 pack of 25
Luer-Lock adapters: 1 pack of 2
5-mL plastic Luer-Lock syringes: 1 pack of 2
1.5-mL microtubes: 6 packs of 100
Spin cartridge extra caps and plugs, 1 pack
of 6 each
Teflon Luer-Lock needles, 1 pack of 10
5188-5231 mRP-C18 High Recovery Protein Fractionation and Desalting Column, see
www.agilent.com/chem/bioreagents for
more details
* Under normal conditions, the kit should last for approximately 200 spin cartridge uses.
For more information or technical assistance, please
call toll free: 1-800-227-9770 or visit our Web site at:
www.agilent.com/chem/bioreagents.
Instructions
26
Instructions
27
©Agilent Technologies, Inc. 2008
Agilent shall not be liable for errors
contained herein or for incidental or
consequential damages in connection
with the furnishing, performance, or
use of this material.
Information, descriptions, and specifications in this publication are subject
to change without notice.
Printed in the USA
Second edition
October 2008
5969-1597